CN109843319A

CN109843319A - Subcutaneous administration of the long-acting factors IX in people

Info

Publication number: CN109843319A
Application number: CN201780042142.6A
Authority: CN
Inventors: C·沃格特; S·普利; J·罗伯特斯; M·诺尔蒂
Original assignee: Kangnobel Linnau Co Ltd
Current assignee: Kangnobel Linnau Co Ltd
Priority date: 2016-07-08
Filing date: 2017-07-07
Publication date: 2019-06-04
Also published as: WO2018007601A1; CA3029887A1; EP3481416A1; KR102446476B1; EP3733199A1; KR20190026863A; DK3481416T3; JP6971301B2; AU2017294555A1; JP2019524089A; ES2832648T3; AU2017294555B2; US20200093901A1; EP3481416B1

Abstract

The present invention relates to the clinically relevant dosage regimens of the long-acting factors IX of subcutaneous administration preventative in human patient (the people FIX merged with human albumin).

Description

Subcutaneous administration of the long-acting factors IX in people

Invention field

The present invention relates to the long-acting factors IX of subcutaneous administration preventative in human patient (the people FIX merged with human albumin) Clinically relevant dosage regimen.

Background of invention

Hemophilia B is x linked recessive hereditary hemorrhagic disease caused by being lacked by plasma thromboplastin component (FIX), blood coagulation because Sub- IX is the core coagulation factor during blood coagulation.The S&S of hemophilia B be it is variable, depend on FIX lack Severity and bleeding position.In the case where most frequently, bleeding be characterized in joint, muscle and soft tissue spontaneity or Bleeding caused by wound.The recurring hemorrhage of same position may cause joint permanent damage.It is rare but threat to life go out Blood is it can also happen that in central nervous system, throat or gastrointestinal tract.

Currently, prevention is considered as the optimal care of severe haemophiliacs, because it reduces the hair of all bleeding episodes Raw rate, and when starting at an early age, also reduce the development of arthropathy.

In the clinical research of intravenous application approach, the long-acting people FIX (" rIX-FP ") merged with human albumin has been shown Show in intravenous application with long half-life period, and the clinical studies show completed goes out fabulous safety and validity. It is using the middle position year spontaneous Bleeding rate (AsBR) that rIX-FP carries out 7 days, 10 days or 14 days preventative-therapeutic subjects 0.00.In < 12 years old children, the overall intermediate value AsBR of each subject is annual 0.00 time after prevention in (7 days) once a week Bleeding.Successful treatment bleeding episode is injected with 1 or 2 rIX-FP；Bleeding episode is in adult's research (research CSL654_3001) 98.6%, bleeding episode is 97.2% in child study (CSL654_3002).This long-acting factors IX is the one of hemophilia management Item significant improvement, because it allows the intravenous application to the less frequency of patient compared with individual FIX.

It is described in US5925739A comprising there is the active Factor IX of at least 200IU/ml or factors IX and increase it The pharmaceutical preparation of the additive of the bioavilability of subcutaneous administration and its use in drug of the preparation for treating hemophilia B On the way, but the data of Factor IX are provided only.In WO2012/006624, the therapeutic chimeric polyeptides comprising FIX are described, It can be merged with FcRn binding partners such as Fc.The document prompt with about once a week or longer interval application at least about 10IU/kg FIXFc is used for prophylactic treatment.The document only discloses the data of FIXFc fusion protein, be in the present invention The different fusion protein of the fusion protein of analysis, and it is with different pharmacokinetic properties, it is therefore desirable to and different gives Prescription case is to maintain certain trough levels of Factor IX activity.

WO2014/052490 is disclosed to be grown for being applied with about 10IU/kg to the dosage of about 200IU/kg to people experimenter The treatment method of factors IX Fc fused polypeptide is imitated, wherein dosing interval is about once a week or longer.WO2015/095925 is public The data of the preventative intravenous administration scheme of long-acting factors IX (including the FIX (" rIX-FP ") connecting with albumin) are opened. The dosage of 25-75IU/kg is described as with interval application at least once a week.It does not disclose about in people in those references The data of the middle long-acting FIX of subcutaneous administration.

Although Liles et al. has delivered the single SC dosage with the FIX in 9.3U/kg blood plasma source to single hemophilia B Report (the Thromb Haemost1997 of patient's administration；77 (5): 944-8), but FIX is subcutaneously injected about to hemophilia B patient Clinical data it is very limited.Preclinical study has shown that biological utilisation degree dramatically different in various animal models According to.For example, preclinical data of the invention shows to depend on animal species, the bioavilability of the rIX-FP of subcutaneous administration is About 13-50%.

The I phase pilot study to the PK of rIX-FP after hemophilia B patient's subcutaneous administration is carried out.Present invention table for the first time Bright, which kind of subcutaneous administration scheme of long-acting FIX can be used in treating the hemophilia B of human patient.Since subcutaneous injection can be by suffering from Person oneself is easily applied in the case where children by the nursing staff in family, therefore the blood that these results allow to significantly improve Friend disease B management, because it allows more convenient and pain less injects FIX.Subcutaneous administration also avoids venous channel (venous Access the risk formed with clot) is polluted in device, otherwise may be hindered some with haemophiliachemophiliac very young youngster Child obtains enough nursing.

Specifically, the present invention relates to the fusion protein (" rIX-FP ") comprising people FIX and human albumin, wherein people FIX By the way that the N-terminal of human albumin, institute can be connected to by participation blood coagulation or by the peptide linker that the protease of activated by thrombin is cut State fusion protein and use the restricted subcutaneous administration scheme of following exemplary: dosing interval is once a day to 10- once a week The dosage of 50IU/kg, preferably dosing interval be once a day, the dosage of every 2 days or every 3 days 25IU/kg.Have shown that these Subcutaneous administration scheme provides enough bioavilabilities, and to maintain in haemophiliac, clinically significant stable state FIX is horizontal.

This progress be improve hemophilia B management important further step because by allow patient easily from My object element IX can be avoided being transfused completely and is injected intravenously.Therefore, present invention reduces the injections due to that may pollute With infusion apparatus and cause infection risk, and by avoid be injected intravenously and be transfused needed for hospital and physician visits come Increase the compliance of patient.The present invention also provides therapeutic choice for the patient with venous channel problem.

Summary of the invention

The present invention provides the clinically relevant dosage regimens for subcutaneous administration rIX-FP.The realization of these dosage regimens is enough Prevent the trough level of the FIX of the bleeding during treatment.Embodiment according to the present invention is described in detail in the claims.

Specifically, the present invention relates to fusion protein, it includes

A) people's factors IX (FIX), and

B) human albumin

It is used in the method for its bleeding for being used to prevent in preventive administration scheme human patient, wherein people FIX passes through peptide The connection of the N-terminal of connector and human albumin, the peptide linker can be cut by participation blood coagulation or by the protease of activated by thrombin, And wherein the fusion protein is waited for the dosage of about 10-50IU/kg, using about once a day between administration about once a week Every subcutaneous administration to the subject.Dosage can be about 10-20IU/kg, about 20-30IU/kg, about 30-40IU/kg, about 40- About 50IU/kg, about 10IU/kg, about 15IU/kg, about 20IU/kg, about 25IU/kg, about 30IU/kg, about 35IU/kg, about 40IU/ Kg, about 45IU/kg or about 50IU/kg.In preferred embodiments, dosage is about 10IU/kg.Preferably implement at another In scheme, dosage is about 20IU/kg.In a further preferred embodiment, dosage is about 25IU/kg.It is preferred at another Embodiment in, dosage 50IU/kg.In in these embodiments any one, dosing interval be can be about daily Once, or about secondary on every Thursdays or 2 days about every or 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.

In a preferred embodiment, dosage is about 10IU/kg, and dosing interval is or about one week about once a day Four times, or 2 days about every, or about every 3 days or about twice a week.

In another preferred embodiment, dosage 25IU/kg, dosing interval be or be about every about once a day Thursday time, or it is 2 days about every or 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.

In another preferred embodiment, dosage 50IU/kg, dosing interval are about once a day, or about weekly Four times, or it is 2 days about every or 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.

In highly preferred embodiment, dosage 25IU/kg, dosing interval is about once a day.It is non-at another In normal preferred embodiment, dosage 25IU/kg, dosing interval is 2 days about every.In another highly preferred embodiment In, dosage 25IU/kg, dosing interval is 3 days about every.

In highly preferred embodiment, dosage is about 25IU/kg, and dosing interval is and blood plasma about once a day In total FIX activity trough level maintain about 8 to about 34IU/dL.In another highly preferred embodiment, dosage is About 25IU/kg, dosing interval are that about once a day, and total FIX activity trough level in blood plasma maintains about 10 to about 30IU/dL.In another highly preferred embodiment, dosage is about 25IU/kg, dosing interval be about once a day, and And total FIX activity trough level in blood plasma maintains about 15 to about 25IU/dL.In another highly preferred embodiment In, dosage is about 25IU/kg, and dosing interval is that about once a day, and total FIX activity trough level in blood plasma maintains about 18 to about 22IU/dL.In another highly preferred embodiment, dosage is about 25IU/kg, and dosing interval is about daily one It is secondary, and total FIX activity trough level in blood plasma maintains about 17IU/dL.

In alternative highly preferred embodiment, dosage is about 25IU/kg, and dosing interval is 2 days about every, and blood Total FIX activity trough level in slurry maintains about 4 to about 17IU/dL.In another highly preferred embodiment, dosage It is about 25IU/kg, dosing interval is 2 days about every, and total FIX activity trough level in blood plasma maintains about 6 to about 15IU/ dL.In another highly preferred embodiment, dosage is about 25IU/kg, and dosing interval is 2 days about every, and in blood plasma Total FIX activity trough level maintain about 7 to about 12IU/dL.In another highly preferred embodiment, dosing interval It is 2 days about every, and total FIX activity trough level in blood plasma maintains about 8IU/dL.

In further highly preferred embodiment, dosage is about 25IU/kg, and dosing interval is 3 days about every, and blood Total FIX activity trough level in slurry maintains about 2.5 to about 12IU/dL.In another highly preferred embodiment, agent Amount is about 25IU/kg, and dosing interval is 3 days about every, and total FIX activity trough level in blood plasma maintains about 3 to about 12IU/dL.In another highly preferred embodiment, dosage is about 25IU/kg, and dosing interval is 3 days about every, and blood Total FIX activity trough level in slurry maintains about 4 to about 11IU/dL.In another highly preferred embodiment, dosage It is about 25IU/kg, dosing interval is 3 days about every, and total FIX activity trough level in blood plasma maintains about 4.5 to about 10IU/dL.In another highly preferred embodiment, dosage is about 25IU/kg, and dosing interval is 3 days about every, and blood Total FIX activity trough level in slurry maintains about 5IU/dL.

In preferred embodiments, the blood plasma level of FIX is maintained above baseline at least about in entire dosing interval 1%, baseline at least about 3% is higher than preferably in entire dosing interval, is higher than baseline at least more preferably in entire dosing interval About 5%, baseline at least about 10% is higher than more preferably in entire dosing interval, and be most preferably higher than in entire dosing interval The trough level of baseline at least about 15%.

In preferred embodiments, connector can be cut by FIXa and/or FVIIa/ tissue factor (TF).Especially excellent In the embodiment of choosing, connector includes the sequence selected from SEQ ID NO:1 and SEQ ID NO:2.

In alternative embodiment, connector has 90% identity with one of SEQ ID NO:1 and SEQ ID NO:2. In another embodiment, with one of SEQ ID NO:1 and SEQ ID NO:2 with 80% identity.At another In embodiment, with one of SEQ ID NO:1 and SEQ ID NO:2 with 70% identity.In another embodiment In, with one of SEQ ID NO:1 and SEQ ID NO:2 with 60% identity.In a further embodiment, with One of SEQ ID NO:1 and SEQ ID NO:2 has 50% identity.

Preferably, fusion protein of the invention has at least 70% identity with sequence shown in SEQ ID NO:3.

SEQ ID NO:3 (mature rIX-FP)

FIX(aa 1-416)

Joint sequence (overstriking and the aa 416-433 underlined)

Albumin sequence (aa 434-1018)

The sequence of fusion protein can have at least 75% identity with sequence shown in SEQ ID NO:3.Merge egg White sequence can have at least 80% identity with sequence shown in SEQ ID NO:3.The sequence of fusion protein can be with Sequence shown in SEQ ID NO:3 has at least 85% identity.The sequence of fusion protein can with shown in SEQ ID NO:3 Sequence have at least 90% identity.The sequence of fusion protein can have at least with sequence shown in SEQ ID NO:3 95% identity.The sequence of fusion protein can have at least 98% identity with sequence shown in SEQ ID NO:3.Melt The sequence of hop protein can have at least 99% identity with sequence shown in SEQ ID NO:3.In preferred embodiment party In case, the sequence of fusion protein has sequence shown in SEQ ID NO:3.

In a preferred embodiment of the invention, human patient suffers from hemophilia B.

For any embodiment of the invention, provide fusion protein for about 100 to 400IU/ml, preferably from about 100, The concentration of 200 or 400IU/ml is applied.Fusion protein can also be provided for applying with the concentration of 600IU/ml or 1200IU/ml With.

The invention also includes the fusion protein of the FIX (more than 3 times of FIX end-stage half-life period) comprising Increased Plasma Half-life, For preventing bleeding in the human patient treated with the FIX of the FIX intravenously applied or the Increased Plasma Half-life intravenously applied It is used in method, wherein the FIX protein of Increased Plasma Half-life waits for subcutaneous administration to subject.On the other hand, the present invention includes packet The fusion protein of FIX (more than 3 times of FIX end-stage half-life period) containing Increased Plasma Half-life, is used to prevent the group of human patient bleeding Treatment is closed, wherein patient is treated with the FIX of the FIX intravenously applied or the Increased Plasma Half-life intravenously applied first, and is being opened Begin fusion protein subcutaneous administration to subject after intravenous FIX treatment.In WO2015/095925 and WO2014/052490 Describe the intravenous application of the FIX of Increased Plasma Half-life.Preferably, fusion protein includes a) factors IX (FIX) and b) half-life period Enhance polypeptide (HLEP).In highly preferred embodiment, fusion protein includes a) people's factors IX (FIX) and b) the white egg of people White, wherein people FIX is by can be by participation blood coagulation or the peptide linker and human albumin that are cut by the protease of activated by thrombin The connection of the end N-.Subcutaneous dosage is preferably with the dosage of about 10-50IU/kg, using about once a day to administration about once a week Interval application.Dosage can be about 10-20IU/kg, about 20-30IU/kg, about 30-40IU/kg, about 40- about 50IU/kg, about 10IU/kg, about 15IU/kg, about 20IU/kg, about 25IU/kg, about 30IU/kg, about 35IU/kg, about 40IU/kg, about 45IU/kg Or about 50IU/kg.In preferred embodiments, dosage is about 10IU/kg.In another preferred embodiment, dosage It is about 20IU/kg.In a further preferred embodiment, dosage is about 25IU/kg.In another preferred embodiment In, dosage 50IU/kg.In in these embodiments any one, dosing interval be can be about once a day, or about every Thursday time, or it is 2 days about every or 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.In highly preferred reality It applies in scheme, dosage 25IU/kg, dosing interval is that about once a day, about every 2 days primary, or about every 3 days primary.Preferably, This method is related to preventive administration scheme.In another highly preferred embodiment, fusion protein includes a) people's factors IX (FIX) and b) part Fc of antibody or a) people's factors IX and b) XTEN.In all above-mentioned fusion proteins, people FIX and HELP can To be directly connected to or be connected by peptide linker.In preferred embodiments, connector can be cut by protease, wherein protease Preferably participate in blood coagulation or the protease by activated by thrombin.On the other hand, the present invention includes for preventing human patient bleeding The people FIX (more than 3 times of FIX end-stage half-life period) of the Increased Plasma Half-life of the conjugation of combined therapy, wherein patient uses vein first The FIX of the FIX of interior application or the Increased Plasma Half-life for the conjugation intravenously applied treatment, and will after starting intravenous FIX treatment The people FIX subcutaneous administration of the Increased Plasma Half-life of conjugation is to subject.Lead to the non-limiting example packet of the conjugation of Increased Plasma Half-life Include Pegylation or either polysialylated.In preferred embodiments, the FIX of the FIX or Increased Plasma Half-life that intravenously apply Application will by the subcutaneous administration of the FIX of subsequent Increased Plasma Half-life replace.In another preferred embodiment, half-life period Extended FIX is applied by intravenous with subcutaneously parallel.

The invention also includes the methods for the bleeding for preventing human patient in preventive administration scheme, including to subject with about Once a day to the fusion protein of dosing interval subcutaneous administration about 10-50IU/kg dosage about once a week, the fusion protein Include

A) people's factors IX (FIX), and

B) human albumin,

Wherein people FIX is connected by the N-terminal of peptide linker and human albumin, the peptide linker can by participation blood coagulation or by The protease of activated by thrombin is cut.Dosage can be about 10-20IU/kg, about 20-30IU/kg, about 30-40IU/kg, about 40- About 50IU/kg, about 10IU/kg, about 15IU/kg, about 20IU/kg, about 25IU/kg, about 30IU/kg, about 35IU/kg, about 40IU/ Kg, about 45IU/kg or about 50IU/kg.In preferred embodiments, dosage is about 10IU/kg.Preferably implement at another In scheme, dosage is about 20IU/kg.In a further preferred embodiment, dosage is about 25IU/kg.It is preferred at another Embodiment in, dosage is about 50IU/kg.In in these embodiments any one, dosing interval can be about every It is primary, or about secondary on every Thursdays or 2 days about every or 3 days about every, or about twice a week, or 5 days about every, or about once a week.

In preferred embodiments, connector can be cut by FIXa and/or FVIIa/ tissue factor (TF).Especially excellent In the embodiment of choosing, connector includes the sequence selected from SEQ ID NO:1 and SEQ ID NO:2.In alternative embodiment, connect One of head and SEQ ID NO:1 and SEQ ID NO:2 have 90% identity.In another embodiment, with SEQ One of ID NO:1 and SEQ ID NO:2 has 80% identity.In yet another embodiment, with SEQ ID NO:1 and One of SEQ ID NO:2 has 70% identity.In yet another embodiment, with SEQ ID NO:1 and SEQ ID One of NO:2 has 60% identity.In a further embodiment, with SEQ ID NO:1 and SEQ ID NO:2 it One has 50% identity.

Preferably, fusion protein of the invention has at least 70% identity with sequence shown in SEQ ID NO:3.Fusion The sequence of albumen can have at least 75% identity with sequence shown in SEQ ID NO:3.The sequence of fusion protein can be with There is at least 80% identity with sequence shown in SEQ ID NO:3.The sequence of fusion protein can be with SEQ ID NO:3 institute The sequence shown has at least 85% identity.The sequence of fusion protein can have extremely with sequence shown in SEQ ID NO:3 Few 90% identity.The sequence of fusion protein can have at least 95% identity with sequence shown in SEQ ID NO:3. The sequence of fusion protein can have at least 98% identity with sequence shown in SEQ ID NO:3.The sequence of fusion protein There can be at least 99% identity with sequence shown in SEQ ID NO:3.In a more preferred embodiment, fusion protein Sequence have SEQ ID NO:3 shown in sequence.

The invention also includes the fusion protein of the FIX (more than 3 times of FIX end-stage half-life period) comprising Increased Plasma Half-life, For preventing bleeding in the human patient treated with the FIX of the FIX intravenously applied or the Increased Plasma Half-life intravenously applied It is used in method, wherein the FIX protein of Increased Plasma Half-life waits for subcutaneous administration to subject.On the other hand, the present invention includes packet The fusion protein of FIX (more than 3 times of FIX end-stage half-life period) containing Increased Plasma Half-life, is used to prevent the group of human patient bleeding Treatment is closed, wherein patient is treated with the FIX of the FIX intravenously applied or the Increased Plasma Half-life intravenously applied first, and is being opened Begin fusion protein subcutaneous administration to subject after intravenous FIX treatment.In WO2015/095925 and WO2014/052490 Describe the intravenous application of the FIX of Increased Plasma Half-life.Preferably, fusion protein includes a) factors IX (FIX) and b) half-life period Enhance polypeptide (HLEP).In highly preferred embodiment, fusion protein includes a) people's factors IX (FIX) and b) the white egg of people White, wherein people FIX is by can be by participation blood coagulation or the peptide linker and human albumin that are cut by the protease of activated by thrombin The connection of the end N-.Subcutaneous dosage is preferably with the dosage of about 10-50IU/kg, using about once a day to administration about once a week Interval application.Dosage can be about 10-20IU/kg, about 20-30IU/kg, about 30-40IU/kg, about 40- about 50IU/kg, about 10IU/kg, about 15IU/kg, about 20IU/kg, about 25IU/kg, about 30IU/kg, about 35IU/kg, about 40IU/kg, about 45IU/kg Or about 50IU/kg.In preferred embodiments, dosage is about 10IU/kg.In another preferred embodiment, dosage It is about 20IU/kg.In a further preferred embodiment, dosage is about 25IU/kg.In another preferred embodiment In, dosage 50IU/kg.In in these embodiments any one, dosing interval be can be about once a day, or about every Thursday time, or it is 2 days about every or 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.In highly preferred reality It applies in scheme, dosage 25IU/kg, dosing interval is that about once a day, about every 2 days primary, or about every 3 days primary.Preferably, This method is related to preventive administration scheme.In another highly preferred embodiment, fusion protein includes a) people's factors IX (FIX) and b) part Fc of antibody or a) people's factors IX and b) XTEN.In all above-mentioned fusion proteins, people's FIX and HELP energy It is enough directly connected to or is connected by peptide linker.In preferred embodiments, connector can be cut by protease, wherein protease Preferably participate in blood coagulation or the protease by activated by thrombin.On the other hand, the present invention includes for preventing human patient bleeding The people FIX (more than 3 times of FIX end-stage half-life period) of the Increased Plasma Half-life of the conjugation of combined therapy, wherein patient uses vein first The FIX of the FIX of interior application or the Increased Plasma Half-life for the conjugation intravenously applied treatment, and will after starting intravenous FIX treatment The people FIX subcutaneous administration of the Increased Plasma Half-life of conjugation is to subject.Lead to the non-limiting example packet of the conjugation of Increased Plasma Half-life Include Pegylation or either polysialylated.In preferred embodiments, the FIX of the FIX or Increased Plasma Half-life that intravenously apply Application will by the subcutaneous administration of the FIX of subsequent Increased Plasma Half-life replace.In another preferred embodiment, half-life period Extended FIX is applied by intravenous with subcutaneously parallel.

Detailed description of the invention

Fig. 1: the blood plasma level (average value ± SD, n=3/ time point) of people FIX antigen (rIX-FP), lineal scale in rabbit.

Fig. 2: the blood plasma level (average value ± SD, n=1-3/ time point) of people FIX antigen (rIX-FP) in pig, it is linear to mark Degree.

Fig. 3: with being averaged for rIX-FP of the dosage level of 125 and 500IU/kg intravenously and after subcutaneous administration to machin Plasma concentration.

Fig. 4: after the dosage level subcutaneous administration to machin of 500IU/kgWith putting down for rIX-FP Equal plasma concentration.

Fig. 5 A: pass through ID (group 1-25IU/kg) subcutaneous dosage in four different human patient (38,39,40 and 50) RIX-FP uncorrected FIX activity relative time figure.

Fig. 5 B: pass through ID (group 2-50IU/kg) subcutaneous agent in five different human patient (38,39,16,17 and 21) The figure of the uncorrected FIX activity relative time of the rIX-FP of amount.

Fig. 6 A: pass through ID (group 1-25IU/kg) subcutaneous dosage in four different human patient (38,39,40 and 50) RIX-FP correction FIX activity relative time figure.FIX active water is corrected for the index decreased of FIX level before being administered It is flat.Once being corrected, any negative value is both configured to 0.01.

Fig. 6 B: subcutaneous by ID (group 2-50IU/kg) in five different people experimenters (38,39,16,17 and 21) The figure of the FIX activity relative time of the correction of the rIX-FP of dosage.It is living for the index decreased correction FIX of FIX level before being administered Property it is horizontal.Once being corrected, any negative value is both configured to 0.01.

Fig. 7: group 3,25IU/kg, every 3 days repeated doses are observed in three different human patient (38,39,40) FIX activity relative time figure.Gray shade is the time interval between the first dosage and last time repeated doses 15.

The simulation drawing of Fig. 8 A: the FIX of daily 25IU/kg active (IU/dL) relative time.

The simulation drawing of active (IU/dL) relative time of Fig. 8 B: every 2 days 25IU/kg FIX.

The simulation drawing of active (IU/dL) relative time of Fig. 8 C: every 3 days 25IU/kg FIX.

Fig. 8 D: with every 3 days of the PK data cover observed after the 15th dosage of application from group 3 (n=3) The simulation drawing of active (IU/dL) relative time of the FIX of 25IU/kg.

Detailed description of the invention

As used herein, " prophylactic treatment ", " preventive administration scheme ", " prevention bleeding (prevention of Bleeding) " or " prevention bleeding (preventing bleeding) " means to apply multi-dose to human patient whithin a period of time Factors IX fusion protein with increase the Factor IX activity in patients blood plasma level.Preferably, increased level is enough to reduce certainly Bleeding of the incidence or prevention of hair property bleeding in the case where unpredictalbe damage.Prophylactic treatment reduces or prevention bleeding Event, for example, described in the on-demand treatment those.Prophylactic treatment can be fixed or can be individuation, such as " give Discussed in medicine interval ", for example, to compensate the changeability between patient.

As used herein, " dosing interval " means the elapsed time amount between the multiple dosage for being applied to human patient.Make It can be the factors IX without albumin of equal quantities (IU/kg) with the dosing interval in the method for the invention of rIX-FP At least about one. five to eight (1.5-8) of dosing interval needed for (that is, the polypeptide being made of the FIX) times.The application present invention Factors IX-albumin fusion protein when dosing interval can be needed for the factors IX without albumin of equal quantities At least about one. five to eight (1.5-8) of dosing interval times.Dosing interval can be equal quantities without albumin it is described because At least about one. five to eight (1.5-8) of dosing interval needed for sub- IX (or the polypeptide being made of the factors IX) times.

As used herein, " median dose " means that the half for studying human patient is used higher than the dosage, and studies people's trouble The half of person is used below the dosage." mean dose (mean dose) " means that average dosage (average dose) (passes through All dosage phase adductions are calculated divided by dose population).For given dose, " about " mean instruction dosage plus or minus 1,2,5,10,15 or the 20% of the dose indicating.Alternatively, " about " means plus or minus 1IU/kg for given dose.For Dosing interval about once a day, " about " mean plus or minus 6 hours.For between about secondary on every Thursdays or every administration in two days Every " about " means plus or minus 8 hours.For about twice a week or every 3 days dosing intervals, " about " mean plus or minus 12 hours.For about every 5 days dosing intervals, " about " means plus or minus 15 hours.For between administration about once a week Every " about " means plus or minus 18 hours.

" total FIX activity trough level " means the active blood plasma level trough level of FIX measured with IU/dL.This can be with Measurement is generated using direct blood coagulation measurement such as aPTT, PT or fibrin ferment to determine.However, further include other measurements, such as using In the chromogenic assay of specific coagulation factor.The example of such measurement or corresponding reagent is using corresponding deficiency of coagulation factors blood plasma (Dade Behring's)SL (aPTT measurement, Dade Behring) orS is (solidifying The measurement of blood zymogen time, Dade Behring), assay kit is generated using the fibrin ferment of such as deficiency of coagulation factors blood plasma (Technoclone, Thrombinoscope), chromogenic assay such as Biophen factors IX (Hyphen BioMed), FVIIa-rTF (Roche Diagnostics GmbH),Factor IX: C/4 (Chromogenix) or other. FIX activity preferably passes through step aPTT measuring method measurement, and wherein Pathromtin SL is used as activator.For total FIX activity paddy Value is horizontal, and " about " means plus or minus 1IU/dL.

" baseline " means preferably to indicate in given patient with IU/dL or live with the FIX that the active % of FIX in Healthy People is indicated Property it is horizontal, be defined as 100IU/dL or 100%.In serious hemophilia B, give patient baseline level be very down to Zero or almost nil, and in moderate hemophilia, the baseline of patient may be higher, for example, higher than 1%, be higher than 2% or be higher than 3% Or it is higher than 4%, and in slight hemophilia, patient baseline could possibly be higher than 5% of FIX active concentration in Healthy People.When according to this Invention application includes i) people's factors IX (FIX) part and ii) fusion protein of human albumin when, FIX active concentration is sharply first Increase and slowly remove, i.e., it returns to individual baseline level.In our current research, baseline level is residual since previous IV is administered Remaining FIX is horizontal and higher, is corrected by subtracting the preceding horizontal index decreased of administration, to reach expected endogenous levels.

" trough level " is the FIX bioactivity described in entire dosage regimen during the treatment for the patient for needing FIX Floor level.Due to changeability between patient, trough level generally means that intermediate value, and it is higher that this means that the research patient of half has The research patient of trough level, half has lower trough level.Although more common using intermediate value, the valley of PK data Level also may be calculated average value, by determining the value phase adduction of all patients divided by patient's number.Trough level is excellent Choosing is higher than 1%, more preferably higher than 2%, even more preferably greater than 3%, even more preferably greater than 4%, even more preferably greater than 5%, also more Preferably higher than 6%, even more preferably greater than 7%, even more preferably greater than 8%, more preferably higher than 9%, even more preferably greater than 10%, also More preferably higher than 11%, even more preferably greater than 12%, even more preferably greater than 13%, even more preferably greater than 14%, it is even higher more Preferably higher than 15%.Further preferred trough level is higher than 20%, is higher than 25%, is higher than 30%, is higher than 35%, is higher than 40%, it is higher than 45%, is higher than 50%, be higher than 55%, be higher than 60%, be higher than 65%, be higher than 70%, be higher than 75%, is higher than 80%, Higher than 85%, it is higher than 90%, is higher than 95%.

" the active blood plasma level of total FIX is maintained into the valley of at least " about " a certain concentration it is meant that in the trouble for needing FIX During a certain dosage regimen of person, total FIX activity in blood plasma is not less than the concentration, wherein total in human normal plasma FIX active concentration is 1IU/ml or 100IU/dL, and wherein it is preferable to use the surveys of a verified step clotting method for FIX activity It is fixed.FIX activity is FIX bioactivity.

Another a certain second concentration of " maintaining the blood plasma level of FIX about " a certain first concentration " peace treaty " " between paddy For value " it is meant that during a certain dosage regimen for the patient for needing FIX, it is dense that the FIX activity in blood plasma is not less than described first Degree, and second concentration is not exceeded, wherein the FIX active concentration in human normal plasma is 1IU/ml or 100IU/dL, And wherein it is preferable to use the measurements of a verified step clotting method for FIX activity.FIX activity is FIX bioactivity.

" about " a certain trough level means average trough level as used herein.For given trough level, " about " mean 1,2,5,10,15,20 or the 30% of shown trough level plus or minus shown trough level.Alternatively, for giving Fixed trough level, " about " means plus or minus 1IU/dL.

Especially in serious hemophilia B, it has to be noted that FIX active concentration is not less than floor level to prevent bleeding. The floor level is known as desired trough level, and the minimum additional levels by endogenous levels plus the FIX of application form.Tight In the hemophilia B patient of weight, when baseline is almost nil, the trough level higher than baseline 1% means that FIX activity is dense in Healthy People About 1% FIX active concentration of degree.The moderate of the active baseline level of the 3%FIX of FIX active concentration in Healthy People In hemophilia B patient, mean that about 4% FIX activity of FIX active concentration in Healthy People is dense higher than the trough level of baseline 1% Degree.

In the present invention, dosing interval can be about once a day, about secondary on every Thursdays, 2 days about every, 3 days about every, about weekly Twice, 5 days about every, or about once a week.This means that interval can be about every 1 to 7 day once, including about every other day, about often Week 5 times, about every 2-4 days primary, and about 3 times a week, about every 4-6 days primary, and about every 5 days primary, and about every 6-8 days primary, 7 days about every Once.Particularly, it is contemplated that daily it is about primary, about secondary on every Thursdays, 2 days about every, 3 days about every, about twice a week, about every 5 days and Dosing interval about once a week.Most preferred dosing interval is that about once a day, every 2 days primary and every 3 days primary.

Alternatively, it is each patient that dosing interval, which can be the other information based on pharmacokinetic data or about the patient, Determining individuation interval.Individuated dose/dosing interval combination can be with those of the fixed intervals scheme in earlier paragraphs It is identical or can be different.The program can be initially at fixed dosing interval, the administration that then it can change as individuation Interval.Alternatively, the program can be initially at fixed dosage (IU/kg) and dosing interval, then it be can change to use The dosing interval of the individuation of fixed dosage.The program can also be initially at fixed dosing interval and dosage (IU/kg), Then it can change as the dosage for the individuation for using the dosing interval of identical fixation.

For about once a day, about 4 times a week, 2 days about every, the about semiweekly dosage regimen of every 3 days peace treaties, it is contemplated that The therapeutic dose of about 10-15IU/kg, about 15-20IU/kg, about 10IU/kg, about 15IU/kg and about 20IU/kg.

About about once a day, 4 times a week or about every 2 days or 3 days about every, about twice a week or about every 5 days peace treaties are every The dosage regimen of Zhou Yici, it is contemplated that about 20-25IU/kg, about 25-30IU/kg, about 30-35IU/kg, about 35-40IU/kg, 40-45IU/kg, about 45-50IU/kg, about 20IU/kg, about 25IU/kg, about 30IU/kg, about 35IU/kg, about 40IU/kg, about The therapeutic dose of 45IU/kg and about 50IU/kg.

Preferred dose and dosing interval of the invention is as follows:

About 10-25IU/kg about once a day, about 10-25IU/kg about 4 times a week, about every 2 days about 10-25IU/ Kg, about every 3 days about 10-25IU/kg, about 10-25IU/kg about biweekly, about 10IU/kg about once a day, about often All 4 about 10IU/kg, about every 2 days about 10IU/kg, about every 3 days about 10IU/kg, about semiweekly about 10IU/kg, About 20IU/kg about once a day, about 20IU/kg about 4 times a week, about every 2 days about 20IU/kg, about every 3 days pacts 20IU/kg, about semiweekly about 20IU/kg, about every 5 days about 20IU/kg, about 20IU/kg about once a week, about daily About 25-50IU/kg, about 25-50IU/kg about 4 times a week, about every 2 days about 25-50IU/kg, about every 3 days about 25- 50IU/kg, about semiweekly about 25-50IU/kg, about every 5 days about 25-50IU/kg, about 25-50IU/ about once a week Kg, about 25IU/kg about once a day, about 25IU/kg about 4 times a week, about every 2 days about 25IU/kg, about every 3 days pacts 25IU/kg, about semiweekly about 25IU/kg, about every 5 days about 25IU/kg, about 25IU/kg about once a week, about daily Primary about 50IU/kg, about 50IU/kg about 4 times a week, about every 2 days about 50IU/kg, about every 3 days about 50IU/kg, about Semiweekly about 50IU/kg, about every 5 days about 50IU/kg, about 50IU/kg about once a week.

" about once a week " includes about every 6 to 8 days one time peace treaty every 7 days primary.

The dosage of 25IU/kg is most preferred dosage, dosing interval be once a day, every 2 days it is primary or every 3 days primary.

As described in Example 1, the preclinical bioavailability data of subcutaneous rIX-FP is widely different between animal model (for example, be respectively 9.29% and 17% for 500IU/kg and 125IU/kg in monkey, and in rabbit, for 500IU/kg and 150IU/kg is respectively 43% and 55%).Clinical data in embodiment 2 shows the long-acting FIX of which kind of subcutaneous dosage for the first time The hemophilia B of human patient can be effectively controlled.

The present invention includes embodiment, wherein after applying 10IU/kg rIX-FP dosage, the active median plasma water of FIX It puts down once a day, 4 times a week, in every 2 days or 3 days every, semiweekly entire dosing interval be maintained above baseline at least about 5% valley.In another embodiment, after applying 25IU/kg or 50IU/kg rIX-FP dosage, during FIX is active Be worth blood plasma level once a day, 4 times a week, every 2 days or 3 days every, twice a week, every 5 days or dosing interval once a week Inside it is maintained above the valley of baseline at least about 5%.

The invention also includes embodiments, wherein 10IU/kg rIX-FP dosage is about applied daily, in entire dosing interval Inside it is maintained above the active median plasma level of FIX of baseline at least about 7%.In another embodiment, wherein about weekly Four application 10IU/kg rIX-FP dosage, be maintained above in entire dosing interval baseline at least about 4% FIX it is active in It is worth blood plasma level.In another embodiment, wherein about every 2 days application 10IU/kg rIX-FP dosage, the active intermediate value of FIX Blood plasma level is maintained above the valley of baseline at least about 3% in entire dosing interval.In a further embodiment, wherein About every 3 days application 10IU/kg rIX-FP dosage, the active median plasma level of FIX are maintained above base in entire dosing interval The valley of line at least about 2%.In another embodiment, wherein being about administered twice weekly 10IU/kg rIX-FP dosage, FIX Active median plasma level is maintained above the valley of baseline at least about 2% in entire dosing interval.Further implementing In scheme, wherein about every 5 days application 10IU/kg rIX-FP dosage, the active median plasma level of FIX is in entire dosing interval Inside it is maintained above the valley of baseline at least about 1.5%.In a further embodiment, wherein about once a week (for example, every 7 It) 10IU/kg rIX-FP dosage is applied, the active median plasma level of FIX is maintained above baseline extremely in entire dosing interval Few about 1% valley.

In another embodiment, wherein about applying 25IU/kg rIX-FP dosage, the active median plasma of FIX daily Level is maintained above the valley of baseline at least about 17% in entire dosing interval.In a further embodiment, wherein about 25IU/kg rIX-FP dosage is applied 4 times a week, and the active median plasma level of FIX is maintained above base in entire dosing interval The valley of line at least about 10%.In a further embodiment, wherein about every 2 days application 25IU/kg rIX-FP dosage, FIX Active median plasma level maintains at least about 8% valley.In a further embodiment, wherein about every 3 days once apply With 25IU/kg rIX-FP dosage, the active median plasma level of FIX maintains at least about 5% valley.Further implementing In scheme, wherein being about administered twice weekly 25IU/kg rIX-FP dosage, the active median plasma level of FIX is between entire administration Every the interior valley for being maintained above baseline at least about 4%.In a further embodiment, wherein about every 5 days applied once 25IU/ Kg rIX-FP dosage, the active median plasma level of FIX maintain at least about 3% valley.In a further embodiment, Wherein (for example, every 7 days) apply 25IU/kg rIX-FP dosage once a week, and the active median plasma level of FIX is entirely being given It is maintained above the valley of baseline at least about 2% in medicine interval.

In a further embodiment, wherein about applying 50IU/kg rIX-FP dosage, the active intermediate value blood of FIX daily Pulp-water puts down the valley for being maintained above baseline at least about 20% in entire dosing interval.In another embodiment, wherein about 50IU/kg rIX-FP dosage is applied 4 times a week, and the active median plasma level of FIX is maintained above base in entire dosing interval The valley of line at least about 12%.In a further embodiment, wherein about every 2 days applied onces 50IU/kg rIX-FP agent Amount, the active median plasma level of FIX maintain at least about 10% valley.In a further embodiment, wherein it is 3 days about every Applied once 50IU/kg rIX-FP dosage, the active median plasma level of FIX maintain at least about 6% valley.At another In embodiment, wherein being about administered twice weekly 50IU/kg rIX-FP dosage, the active median plasma level of FIX is entirely being given It is maintained above the valley of baseline at least about 5% in medicine interval.In a further embodiment, wherein about every 5 days applied onces 50IU/kg rIX-FP dosage, the active median plasma level of FIX maintain at least about 4% valley.In another embodiment In, wherein (for example, 7 days) apply 50IU/kg rIX-FP dosage about once a week, the active median plasma level of FIX is entire It is maintained above the valley of baseline at least about 2% in dosing interval.

Alternatively, in these embodiments, the active blood plasma level of FIX may be calculated average value.

Particularly, the present invention provides the preventive administration schemes comprising people FIX and the fusion protein of human albumin, wherein People's factors IX (FIX) is partially connect with human albumin, and wherein people FIX is by can be by participation blood coagulation or by the egg of activated by thrombin The peptide linker that white digestion is cut is connected to the N-terminal of human albumin, and wherein fusion protein is waited for the dosage of about 10-50IU/kg, used About once a day to dosing interval subcutaneous administration about once a week to subject.

With allowing the rIX-FP fusion protein of connector cut before FIX activation to show the increasing due to caused by albumin The half-life period of the FIX added, but occur before bleeding episode due to cutting, the half-life period of the FIX of activation is before activation It reduces.On the other hand, if FIX activation after cut, albumin increase FIX half-life period, but activation FIX still with Albumin fusion, therefore its activity is low.

In the sense of the present invention, the proteolysis cutting in blood coagulation associative mode be due at least one coagulation factor or The activation of blood coagulation confactor and occur any proteolysis cutting.Almost live in parallel with the cutting of the proteolysis of joint peptide Change coagulation factor.Activation can be for example by the proteolysis cutting of coagulation factor or by combining co-factor.Albumin Increase the half-life period of FIX in blood until bleeding episode occurs, bleeding episode activates FIX simultaneously and cuts FIX from albumin.It cuts The steric hindrance for damaging polypeptide from any receptor 1 activity as caused by albumin is cut, so that the height for allowing to generate reservation FIX rubs The fusion protein of your specific activity.Then, due to the loss of albumin, the FIX of cutting is removed from blood rapidly.It cannot be cut with it The counterpart cut is compared, and such fusion protein shows improved half-life period and increased molar specific activity.Therefore, with comprising The fusion protein that cannot be cut of rIX is compared, and less rIX-FP is needed to provide therapeutic effect.

Preferred fusion protein according to the present invention is at least one blood coagulation related assays and by with amino acid The molar specific activity of the Therapeutic fusion proteins that cannot cut connector connection of sequence GGGGGGV (SEQ ID NO:4) is (especially Molar ratio blood coagulation related activity) it compares, there is the molar specific activity for the Therapeutic fusion proteins for increasing at least 25% (especially to rub You are than blood coagulation related activity) those of fusion protein.More preferably at least one available different blood coagulation related assays The fusion protein of molar specific activity increase at least 50%, it is even furthermore preferable that molar specific activity increase at least those of 100% Fusion protein.

In a further embodiment, joint peptide includes the cleavage site of more than one protease.This can pass through energy Enough joint peptides cut by different protease in same position or by providing the connector of two or more difference cleavage sites Peptide is realized.There may be such vantages, and wherein Therapeutic fusion proteins must be activated by proteolysis cutting To realize enzymatic activity, and wherein different protease can contribute to the activation step.The activation of FIX can by FXIa or FVIIa/ tissue factor (TF) is realized.In preferred embodiments, connector can be by FIXa and/or FVIIa/ tissue factor (TF) it cuts.

RIX-FP (rFIX- albumin fusion protein) is described as with shown in SEQ ID NO:3 in WO2015/095925 Sequence.Such as pass through 5.3 times of longer half-life period (t_1/2), 7 times reduction CL and 7 times of higher AUC shown by, it with RFIX (for example,) compared to extended plasma circulation.In addition, when the pharmacokinetics ginseng for comparing rIX-FP Number withIn FDA prescription information when the corresponding pharmacokinetic data of FIX-Fc, with rIX-FcIt compares, rIX-FP has the CL/BW, about of about 3.8 times of higher AUC0-inf, about 3.7 times of reductions The 26% higher increment rate of recovery, about 3.2 times of less volume of distribution and the drug mean residence time for increasing about 19%.

Prevention is to prevent the treatment of expected bleeding by injection factor concentrate.According to coagulation factor level > 1IU/ The observation that the moderate haemophiliac of dl (> 1%) seldom undergoes hematostaxis and maintains with much better function of joint As a result think to realize prevention.Therefore, FIX activity is maintained higher than 1% to prevent the prevention of bleeding and destruction of joint from should be Maintain normal muscle skeletal function therapeutic purpose (GUIDELINES FOR THE MANAGEMENT OF HEMOPHILIA, the 2 editions, Treatment Guidelines Working Group represents World Federation of Hemophilia (WFH) It formulates).

The present invention relates to the effective subcutaneous administration schemes of the clinic of the fusion protein comprising people FIX and human albumin, use It is used in the method for bleeding for preventing human patient in preventive administration scheme.It is pre- that these discoveries further improve hemophilia Anti- property treatment does not need hospital or physician visits because it, which allows haemophiliac, self applies FIX.

In preferred embodiments, the blood plasma level of FIX is maintained higher than baseline at least about in entire dosing interval 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 7%, at least about 8%, At least about 10%, at least about 12%, at least about 15%, at least about 18%, at least about 20%, or at least about 30% valley, it is excellent It is selected in the valley maintained in entire dosing interval higher than baseline 8 to 34% or 4 to 17% or 2.5 to 12%.

People FIX

People FIX is a member in vitamin k dependent polypeptides group, is the single chain glycoprotein that molecular weight is 57kDa, Its as 415 amino acid inactive proenzyme by hepatocytes secrete into blood flow.It contains the end the N- Gla knot positioned at polypeptide 12 γ-carboxyl-glutaminic acid residues in structure domain.Gla residue needs vitamin K just to can be carried out biosynthesis.Gla structural domain it Afterwards, there are two epidermal growth factor domains, an activated peptide and a trypsase type serine protease domains. The further posttranslational modification of FIX includes hydroxylating (Asp 64), and N-type glycosylates (Asn157 and Asn167) and O-type sugar Base (Ser53, Ser61, Thr159, Thr169 and Thr172), sulphation (Tyr155) and phosphorylation (Ser158).

By proteolysis of the activated peptide at Arg145-Ala146 and Arg180-Val181, its work is converted by FIX Property form (factors IX a), results in two polypeptide chains, N- terminal light chain (18kDa) and C- terminal heavy chain (28kDa), by One disulfide-bridged, two is connected together.The activation cutting of factors IX can be for example real by factor XI, plasma thromboplastin antecedent a or factor VIIa/TF in vitro It is existing.Factors IX is present in human plasma with the concentration of 5-10 μ g/ml.It was found that terminal plasma half-life of the factors IX in people is about 15 to 18 hours (White GC et al. 1997.Thromb Haemost.78:261-265；Ewenstein BM et al. 2002.Pharmacokinetic analysis of plasma-derived and recombinant F IX concentrates in previously treated patients with moderate or severe hemophilia B.Transfusion 42:190-197)。

Human albumin

Albumin, albumin family member and immunoglobulin and its segment or derivative is hereinbefore described as half Decline the phase enhancing polypeptide (HLEP) example.Term " human serum albumins " (HSA) and " human albumin " (HA) in this application may be used It is used interchangeably.

As used herein, " albumin " is referred to as albumin polypeptide or amino acid sequence or albumin fragment or variant, One or more functional activities (such as bioactivity) with albumin.Particularly, " albumin " mean human albumin or its The human albumin of segment, especially mature form.For example, albumin can have as (it passes through reference to US2008260755A1 Be integrally incorporated herein) described in sequence or its variant.The Albumin in Partial of albumin fusion protein may include overall length HA sequence Column, or may include that can stablize or its active one or more segment of extended treatment.The length of such segment can be 10 A or more amino acid, or may include about 15,20,25,30,50 or more the continuous amino acids from HA sequence, or It may include some or all of HA specific domain.

The Albumin in Partial of albumin fusion protein of the invention can be the variant of natural or artificial normal HA.This hair The therapeutical peptide part of bright fusion protein is also possible to the variant of corresponding treatment polypeptide as described herein.Term " becomes Body " includes insertion, missing and replaces, no matter its be it is conservative or non-conservative, it is natural or artificial, wherein this variation base Do not change the active site or active structure domain for assigning the therapeutic activity of therapeutical peptide in sheet, is such as integrally incorporated by reference Described in the US2008260755A1 of this paper.

The present invention is more particularly directed to fusion proteins, and it includes a) people's factors IXs (FIX) and b) human albumin, and wherein people FIX is logical The end N- for crossing peptide linker and human albumin connects, and the peptide linker can be by participation blood coagulation or by the protease of activated by thrombin Cutting.With not with albumin connection coagulation factor compared with, the fusion protein have increased Half-life in vivo, and with it is corresponding Have and cannot cut the fusion protein of connector and compare, at least one available different blood coagulation related assays, the fusion egg It is white that there is at least 25% higher molar specific activity.

" people's factors IX " in above-mentioned definition includes polypeptide (including any natural polymorphic with natural acid sequence Property).It further includes with the polypeptide of amino acid sequence slightly modified, it may for example comprise terminal amino acid deletions or addition are repaired The end N- or the end C- of decorations, as long as these polypeptides substantially retain the activity of corresponding treatment polypeptide.Including variant one It is different from wild-type sequence in a or more amino acid.This different example may include the end N- and/or the end C- The truncation of one or more amino acid residues (such as preferably 1 to 30 amino acid residue), or add in the end N- and/or the end C- One or more additional residues and conserved amino acid are added to replace, i.e., what is carried out in the amino acid group with similar features takes Generation, such as (1) p1 amino acid, (2) acidic amino acid, (3) polar amino acid, (4) basic amino acid, (5) hydrophobic amino acid, (6) aromatic amino acid.The example of this conservative substitution is as shown in the table.

Table 1: the conservative substitution of amino acid

The Half-life in vivo (being normally identified as end-stage half-life period or β-half-life period) of fusion protein of the invention is usually than non- The Half-life in vivo of fused polypeptide up at least about 25%, preferably at least about 50%, more preferably above 100%.

Compared with the corresponding fusion protein of no connector that can be cut, fusion protein of the invention is excellent at least 25% Choosing at least 50%, more preferably at least 100% increased molar specific activity.

In this respect, molar specific activity (or especially it is considered herein that molar ratio blood coagulation related activity) is defined as every mole Activity expressed by (or such as nmole) interested therapeutical peptide or Therapeutic fusion proteins.The calculating of molar specific activity Allow directly than the activity of the different constructs less influenced by the different molecular weight or optical density of studied polypeptide.Molar ratio is living Property can be as calculated in the following table 2 exemplified by FIX and rIX-FP fusion protein.

Table 2: the calculating of the molar specific activity as shown in the FIX-HSA fusion protein purified

1) R.G.Di Scipio et al., Biochem.16:6698-706 (1977)

2) (3) C.Chaudhury et al. J.Exp.Med.197: 315-322 (2003)

In order to determine molar ratio blood coagulation related activity, it can be used and determine enzyme relevant to coagulation process or co-factor activities Any measurement.

Therefore, " blood coagulation related assays " in the sense of the present invention are to determine enzyme relevant to coagulation process or co-factor activities Any measurement, or can determine any measurement that endogenous or extrinsic clotting cascade have been activated.Therefore, " blood coagulation is related " Measurement can be direct blood coagulation measurement, as aPTT, PT or fibrin ferment generate measurement.However, further include other measurements, such as using In the chromogenic assay of specific coagulation factor.The example of such measurement or corresponding reagent is using corresponding deficiency of coagulation factors blood plasma (Dade Behring's)(aPTT measurement, Dade Behring) or (prothrombin time, Dade Behring) generates measurement reagent using the fibrin ferment of such as deficiency of coagulation factors blood plasma Box (Technoclone, Thrombinoscope), chromogenic assay such as Biophen factors IX (Hyphen BioMed),FVIIa-rTF (Roche Diagnostics GmbH),Factor IX: C/4 (Chromogenix) or it is other.

For purposes of the present invention, it is believed that the increase in above-mentioned any measurement or equivalent blood coagulation related assays is shown The increase of molar specific activity.For example, 25% increase refers in any of above or equivalent measurement 25% increase.

In order to determine whether Therapeutic fusion proteins fall into the scope of the present invention, compare the molar specific activity of these protein Standard be that wherein corresponding coagulation factor and human albumin pass through with amino acid sequence GGGGGGV (SEQ ID NO:4) The construct for the connector connection that cannot be cut.

For FIX, aPTT measurement is commonly used in measurement blood coagulation activity.However, it is possible to using other blood coagulation related assays or Measuring principle determines the molar specific activity of FIX.

While it is desirable to non-activated coagulation factors to have the high internal rate of recovery and long half-life period, but it is advantageous that limit Coagulation factor processed risk before the half-life period after its activation or the activation of its co-factor is to avoid thrombus.Therefore, starting blood coagulation Cheng Hou should reduce the half-life period of factor Xa again.This can be by enhancing the inactivation in blood coagulation associative mode or passing through Coagulation factor is eliminated to realize.

According to the present invention inactivate means that the activity of therapeutical peptide reduces, can be for example by coagulation factor and corresponding The compound of the inhibitor of coagulation factor is formed or by known further proteolysis cutting (such as FVIII's and FV In the case of) cause.

The deactivation rate of the Therapeutic fusion proteins of activation be defined as activity decline (such as by react with inhibitor or lead to Cross proteolytic inactivation) rate.Deactivation rate can be by there are the inhibitor of this of physiological amount coagulation factor the case where Under at any time follow the molar specific activity of the coagulation factor of activation to measure.

Alternatively, can be after the product of activation be applied to animal, then using activity and antigen measuring appropriate Time range tests plasma sample to determine deactivation rate.

When for Therapeutic fusion proteins, it is thus necessary to determine that when whether these protein fall into the scope of the present invention, with these The standard that the deactivation rate of therapeutic protein compares is that wherein corresponding coagulation factor and human albumin pass through with amino The construct that the connector that cannot be cut of acid sequence GGGGGGV (SEQ ID NO:4) combines.

The supersession rate of the Therapeutic fusion proteins of activation is defined as the rate that polypeptide is eliminated from the circulation of human or animal. Supersession rate can be determined by the pharmacokinetics of the Therapeutic fusion proteins of measurement subcutaneous administration post activation.By using anti- Original measurement, can determine the elimination by directly removing from circulation.Can deactivation rate in addition be determined using determination of activity.

When for Therapeutic fusion proteins, it is thus necessary to determine that when whether these protein fall into the scope of the present invention, with these The standard that the supersession rate of therapeutic protein compares is that wherein corresponding coagulation factor and human albumin pass through with amino The construct that the connector that cannot be cut of acid sequence GGGGGGV (SEQ ID NO:4) combines.

According to the present invention, fusion protein includes by the people FIX of the end N- of peptide linker and human albumin connection, the peptide Connector can be cut by participation blood coagulation or by the protease of activated by thrombin.Connector should be non-immunogenic and should have There are enough flexibilities to allow to be cut by protease.

The connector that can be cut is preferably comprised derived from sequence of therapeutical peptide a) to be administered itself (if it contains During the activation of therapeutical peptide proteolysis cut proteolytic cleavage sites), b) therapeutical peptide substrate polypeptide, Or substrate polypeptide c) cut by protease, the protease pass through the direct or indirect participation activation of therapeutical peptide or shape At.

Connector area in further preferred embodiment includes the sequence of therapeutical peptide to be applied, should lead to expression The risk of the neoantigen property of fusion protein reduces.

In preferred embodiments, the sequence of region of activation of the joint sequence from FIX, any substrate from FIX is such as The cutting area of FX or FVII, or from by the cutting of any substrate polypeptide of protease (it activates FIXa and participates) cutting Area.

In highly preferred embodiment, joint peptide is derived from FIX itself.In a further preferred embodiment, Joint peptide is derived from FX or FVII.In embodiment still more preferably, joint sequence include two can by FXIa or The cutting sequence of FVIIa/TF (the relevant FIX activator of two kinds of physiology) cutting.

The variant and segment of the connector are also included in the present invention, if connector still be able to be cut one kind of connector or Multiple protein digestion is cut.Term " variant " includes conservative or non-conservative insertion, missing and substitution.

Pharmaceutical composition and method of application

Fusion protein of the invention can be incorporated into the pharmaceutical composition of suitable subcutaneous administration.Such composition usually wraps Containing protein and pharmaceutically acceptable carrier.As used herein, " pharmaceutically acceptable carrier " is intended to include and pharmacy skin Lower application compatible any and all solvents, decentralized medium, coating, antibacterial agent and antifungal agent, isotonic agent and absorption delay Agent etc..Solution or suspension for subcutaneous application can include following components: sterile diluent, such as water for injection, salt are molten Liquid, expressed oi, polyethylene glycol, glycerol, propylene glycol or other synthetics；Antibacterial agent such as benzyl alcohol or P-hydroxybenzoic acid Methyl esters；Antioxidant, such as ascorbic acid or sodium hydrogensulfite；Chelating agent such as ethylenediamine tetra-acetic acid (EDTA)；Buffer such as acetic acid Salt, citrate or phosphate and tonicity contributor, such as sodium chloride or glucose.Suitable carrier is described in latest edition Remington's Pharmaceutical Sciences (the canonical reference text of this field is incorporated by reference into this Text) in.The preferred embodiment of such carrier or diluent includes but is not limited to water, salt water, Ringer's solution, dextrose solution and 5% human serum albumins.Also liposome and non-aqueous vehicles such as expressed oi can be used.These media and reagent are used for medicine The purposes of active substances is well known in the art.Unless any conventional media or reagent and reactive compound not phase Hold, otherwise considers to use them in the composition.The reactive compound of supplement can be also incorporated into composition.Can with acid or Alkali such as hydrochloric acid or sodium hydroxide adjust pH.Parenteral administration can be encapsulated in the ampoule being made of plastic or glass, disposable note In emitter or multiple dose vials.It is preferred administration method as hypodermic application.

It include sterile aqueous solution (when water can be dissolved in) or dispersion and use suitable for injecting the pharmaceutical composition used The aseptic powdery of solution or dispersion is injected in extemporaneous preparation of sterile.For subcutaneous administration, suitable carrier includes physiological saline, Bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS) (PBS).In all cases, Composition must be sterile and should be mobility so as in the presence of the ability for being easy injection.It must manufacture and store Under the conditions of stablize, and must be prevented from the contamination of microorganism such as bacterium and fungi.Carrier can be solvent or decentralized medium, It contains such as water, ethyl alcohol, polyalcohol (such as glycerol, propylene glycol and liquid macrogol) and its suitable mixture.Example Such as by using coating such as lecithin, by maintaining required granularity in the case of a dispersion and by using surface-active Agent is able to maintain that mobility appropriate.By various antibacterial agents and antifungal agent, for example, p-hydroxybenzoate, methaform, Phenol, ascorbic acid, thimerosal etc. can be realized the effect for preventing microorganism.In many cases, include in the composition etc. Agent is opened, such as sugar, polyalcohol such as mannitol, D-sorbite, sodium chloride will be preferred.By being inhaled in the composition comprising delay The reagent of receipts, such as aluminum monostearate and gelatin, the extension that can be realized injectable composition absorb.

Aseptic injectable solution can by by the desired amount of reactive compound (such as rIX-FP) as needed with arrange above One of ingredient of act or combination are incorporated into solvent appropriate, and then prepared by filtration sterilization.In general, by that will activate It closes in object incorporation sterile carrier and prepares dispersion, the sterile carrier contains basic dispersion medium and required from upper The other compositions of ingredient those of are enumerated in face.In the case where being used to prepare the aseptic powdery of aseptic injectable solution, preparation method Be vacuum drying and freeze-drying, generate active constituent add needed for any other of its previous sterilefiltered solutions at The powder divided.

Injection preparation can factors IX (FIX) fusion protein (such as rIX-FP) containing therapeutically effective amount, which can It is determined by technical staff.Particularly, factors IX (FIX) fusion protein (i.e. rIX-FP) can with about 100 to 2000IU/ml it is dense Degree application.For example, fusion protein can be provided for about 100 to 400IU/ml, or about 100,200 or 400IU/ml's is dense Degree application.Fusion protein can also be provided for about 600IU/ml, 800IU/ml, 1000IU/ml, 1200IU/ml, 1400IU/ml, 1600IU/ml, 1800IU/ml or 2000IU/ml application.Fusion protein can also be provided for about 2500IU/ml, 3000IU/ml, 3500IU/ml or 4000IU/ml application.

Particularly advantageously with dosage unit form compounding pharmaceutical composition, such as composition for injection, in order to apply With the uniformity of dosage.Dosage unit form used herein, which refers to, is suitable as the unit dose of patient to be treated physically Discrete unit；Each unit contains the reactive compound of predetermined amount, and being computed can generate together with required pharmaceutical carrier Required therapeutic effect.Agent is determined and depended directly on by the specific characteristic and the particular treatment effect to be realized of reactive compound Measure the specification of unit form.

Pharmaceutical composition can be included in container, packaging or distributor together with application specification.

The present invention will be more clearly understood by reference to following embodiment, these embodiments purpose only for illustrative purposes And it is included herein, it is not intended to the limitation present invention.The all patents and publications being mentioned above is explicitly by introducing It is incorporated herein.

Embodiment

Embodiment 1- preclinical study

A. single SC or intravenous injection rabbit after rIX-FP andPharmacokinetics

The purpose of the research be study subcutaneous administration to rabbit after rIX-FP bioavilability, and further with commercially available blood Starch Human factor IX (FIX) product in source (i.e.P) more subcutaneous pharmacokinetic curve.

Four groups of rabbits (every group includes 3 jennies) receive 150IU/kg (rIX-FP) and 500IU/kg (rIX-FP respectively WithP) single of single SC (s.c.) dosage of dosage level or 150IU/kg (rIX-FP) dosage level is quiet (i.v.) dosage in arteries and veins.Hereafter, blood sample is extracted to be up to 10 days.Rabbit blood is carried out using enzyme linked immunosorbent assay (ELISA) (ELISA) technology The subsequent measurements of people FIX antigen concentration in slurry samples.

The results of the study show that rIX-FP is transported to vascular system after subcutaneous injection in rabbit.It is applied in s.c. Observe that the dose dependent of people's FIX blood plasma level increases after 150 and 500IU/kg rIX-FP, 24 and 32 is small upon administration When between reach peak plasma level (Fig. 1).Therefore, s.c. is using the respective bioavilability of rear rIX-FP for 150IU/kg It is 55%, is 43% (48%=geometrical mean) for 500IU/kg group.WithS.c. using comparing, People's FIX blood plasma level after the s.c. application of 500IU/kg rIX-FP is higher and more slowly reduces.Therefore, s.c.rIX-FP Administration (1.01IU/mL) after most adult FIX blood plasma level beAbout 6 times of (0.17IU/mL) after P administration, Peak plasma level is had reached after about 10 hours.In addition, the AUC after s.c. application rIX-FP isAbout the 13 of P Again (table 3).I.v. or s.c. application rIX-FP orThe adverse reaction for the treatment of is not observed after P.

Table 3: the geometrical mean of the pharmacokinetic parameter estimation of rabbit

Parameter is estimated by non-compartment method,

F=bioavilability

In short, the research have shown that by rIX-FP subcutaneous administration after rabbit bioavilability be 43-55%.RIX-FP's S.c. the tolerance applied is good, has the relevant bioavilability of pharmacology, and total exposure is come better than commercially available blood plasma The FIX product-in source

B.i.v. the pharmacokinetics of CSL654 (rIX-FP) after pig is applied to respect to s.c.

Object of this investigation is to study the Pharmacokinetic Characteristics (packet that rIX-FP after intravenous and subcutaneous administration is carried out to pig Include subcutaneous bioavilability).

Two groups of pigs (every group includes 3 bucks) receive in single dose intravenous the 250IU/kg rIX-FP of (i.v.) dosage Or the 241.5IU/kg rIX-FP of single SC (s.c.) dosage.Hereafter, it is based on using enzyme linked immunosorbent assay (ELISA) (ELISA) Method measures FIX antigen concentration, tracks plasma thromboplastin component (FIX) upon administration blood plasma level 17 days.

Be applied to the rIX-FP of pig be it is well tolerable, i.e. the secondary for the treatment of is not observed after i.v. or s.c. application and makees With.

After s.c. applies rIX-FP, peak F IX blood plasma level (Fig. 2, table 4) is reached after about 22 hours.S.c. it answers It is 22% with the respective bioavilability of rear rIX-FP.After s.c. (53h) and i.v. (44h) administration, end-stage half-life period is phase When.

Table 4: the geometrical mean of pig pharmacokinetic parameters estimation

Parameter is estimated by non-compartment method.

Pharmacokinetic show s.c. application rIX-FP after bioavilability be 22%, and end-stage half-life period with it is quiet Administration is suitable in arteries and veins.

C.i.v. rIX- after primate is applied to respect to s.c.Pharmacokinetics

This research is designed to assess the medicine generation in single dose intravenous with rIX-FP after subcutaneous administration to male and female cynomolgus monkeys Dynamics.It is a kind of commercial product and is used as with reference to test item.

Seven groups of machins (every group includes a buck and a jenny) receive in single dose intravenous or subcutaneous agent The rIX-FP of amount orDosage level range is 125 IU/kg to 500 IU/kg.From every within 10 days time Blood sampling in monkey.Since preparation prepares incorrect in the 1st, 2 and 4 group, the 3rd, 5,6 and 7 group of blood plasma sample is only analyzed Product.

The ELISA method of use experience card carries out the measurement of people's factors IX concentration in monkey plasma sample.

Apply rIX-FP orThe hard to bear property in part is not observed afterwards.

Intravenous application rIX-FP and subcutaneous administration rIX-FP andPharmacokinetics concentration data afterwards is Variable, and due to the height of the horizontal castle's intrinsic factor IX level between individual animals of the castle's intrinsic factor IX detected in sample Changeability is difficult to determine when that realization returns to background level.Therefore, these data are not suitable for complete pharmacokinetic data point Analysis.In order to consider the Endogenous level of factors IX, by the concentration before administration being subtracted from the measurement concentration of each time point come school The plasma concentration of the factors IX of just every monkey.The value of these baseline corrections for calculate rIX-FP andCmax With the bioavilability of AUCt value and rIX-FP.

After intravenous and subcutaneous administration rIX-FP withMean plasma concentration-time graph be shown in Fig. 3 In 4.It is listed in Table 5 below by the pharmacokinetic parameter that these data obtain.

Table 5: the mean serum pharmacokinetic parameter in machin

In short, it may be concluded that the application of research drug has good tolerance, after subcutaneous administration rIX FP Cmax and AUCt value is higher than applicationRespective value afterwards, and after subcutaneous administration rIX-FP, in 125 IU/kg It is respectively 17.0% and 9.29% (average value: 13%) with the bioavilability under the dosage level of 500 IU/kg.

D. after subcutaneous administration to factor Ⅸ deficient mouse rIX-FP bioavilability

The purpose of this research is the bioavilability of rIX-FP after studying subcutaneous administration to factor Ⅸ deficient mouse, and further Compare its subcutaneous Pharmacokinetic Characteristics and commercially available recombinant human blood coagulation factor IX (FIX) product (i.e.) skin Lower Pharmacokinetic Characteristics.

Five groups of factor Ⅸ deficient mouses (every group comprising 12-15 only female or bucks) acceptable dose levels are respectively 125IU/kg (rIX-FP) and 250IU/kg (rIX-FP and) single SC (s.c.) dosage or dosage be (i.v.) dosage in the single dose intravenous of 125IU/kg (rIX-FP).Hereafter, blood sample is extracted to be up to four days.Then measured using blood coagulation Method and enzyme-linked immunosorbent assay (ELISA) carry out the measurement of people FIX activity and antigen concentration in mouse plasma sample respectively.

In a part of this research, the s.c. application of rIX-FP is compared with respective i.v. administration.It is studying Second part, compare two difference s.c. dosage of rIX-FP, at the same in addition by maximum dose level with it is isodoseIt compares.

During research i.v. or s.c. application rIX-FP orTreatment is not observed locally or systemically afterwards Ill-effect.

The research the results show that in factor Ⅸ deficient mouse, rIX-FP is transported to vascular system after subcutaneous injection.

Study part 1: 16 hours (activity levels) and 24 small upon administration after s.c. applies 125IU/kg rIX-FP When (antigen levels) between reach peak plasma level (table 6 and 7).Respective bioavilability is FIX activity level 50% and FIX Antigen levels 31%.

The difference for the PK value derived from activity and antigen FIX level observed may be the difference due to measurement sensitivity (measurement sensitivity of FIX determination of activity is higher compared with using the antigen of ELISA to measure).

It studies part 2: after s.c. applies 125 and 250IU/kg rIX-FP, using activity or antigen FIX blood plasma Level is for estimating, pharmacokinetics and dose proportional (table 6 and 7).Reach Peak plasma between 16 to 24 hours again It is horizontal.

It is applied with s.c.It compares, s.c. applies 250IU/kg rIX-FP descendant FIX active blood plasma levels Higher and more slowly reduction (table 6).Therefore, the maximum blood plasma level (0.69IU/mL) of rIX-FP is About 7 times of (0.10IU/mL).In addition, the AUC (38.6h*IU/mL) after s.c. application rIX-FP is(4.6h* IU/mL) about 8 times.Based on FIX antigen levels (table 7), the maximum FIX blood plasma level (0.54IU/mL) after rIX-FP administration isAbout 5 times of (0.11IU/mL) after administration, and the AUC (26.0h*IU/mL) after s.c. applies rIX-FP isAbout 15 times of (1.7h*IU/mL).

Table 6: the estimation of factor Ⅸ deficient mouse pharmacokinetic parameters (FIX activity)

Parameter is estimated by non-compartment method

N.c.=is not calculated

Table 7: the estimation of factor Ⅸ deficient mouse pharmacokinetic parameters (people FIX antigen)

Parameter is estimated by non-compartment method

In short, s.c. application rIX-FP have good tolerance, have the relevant bioavilability of pharmacology and Pharmacokinetics is better than commercially available recombination FIX product

Embodiment 2- clinical research

The bioavilability of rIX-FP and dosage simulation after subcutaneous administration to hemophilia B patient

The I phase pharmacokinetic of subcutaneous administration rIX-FP is carried out as non-blind, single increment dose study, with assessment Subcutaneously (s.c.) applies after 25 or 50IU/kg rIX-FP rIX-FP in hemophilia B subject (respectively group 1 and group 2) Pharmacokinetics and safety.Also have evaluated the repeated doses application (group 3) of every 25IU/kg for giving 15 times for 3 days.

It is male, at least 18 years old, weight 50.0 that the main eligibility criteria of subject, which is subject, in subcutaneous subgroup research To between 100.0kg, participation at present studies CSL654_3003 and with hemophilia B (FIX activity≤2%), and has received rIX-FP≥100ED.The bleeding episode that threat to life is lived through during 3 months or the subject for carrying out major operation are not eligible for Carry out the research.

The subject for participating in subgroup research is sequentially allocated group, and (group 1 [single 25IU/kg dosage], group 2 are [single A 50IU/kg dosage] or group 3 [repeated doses of 25IU/kg are applied]).

Single dose analyzes (25IU/kg and 50IU/kg)

For group 1 and group 2, the rIX-FP of single dose is applied at least 14 days after rIX-FP application previous, and Before administration, apply after 30 minutes, 24,48,72,120,168,240 and 336 hours collections blood samples for PK analysis.? After 15th day 336 hour time point collected last SC PK sample, the 50IU/kg rIX-FP of single IV dosage is applied, and Blood sample is collected at selected time point to analyze for PK.PK is calculated using FIX activity, passes through central laboratory (CSL Behring the step aPTT measuring method measurement in), and use Pathromtin SL as activator.

As the end that subgroup research is gone to a doctor, return to assess safety within subject (the 28th day) within 2 weeks.At the 28th day Subgroup research it is medical after, subject restores its IV rIX-FP prevention scheme in main research.

The pharmacokinetics of rIX-FP after measurement assessment subcutaneous administration based on FIX activity level in blood plasma.Fig. 5 A and 5B When showing that in each human patient, not corrected FIX activity is opposite at the rIX-FP of (25 and 50IU/kg) subcutaneous dosage Between figure.FIX activity level is corrected for the index decreased of FIX level before being administered, and is expected especially because baseline is higher than Endogenous levels (due to the fact patient is from previous intravenous administration research).Once it is corrected, x negative value is (total Have 4 in totally 30 PK sample spots) be set as 0.01% threshold value enable to carry out models fitting.Fig. 6 A and 6B show people Pass through the figure of the corrected FIX activity relative time of the ID of the rIX-FP of 25 and 50IU/kg subcutaneous dosage in patient.These figures It is horizontal before height administration caused by being left before display SC application by IV, and blood plasma level increases range height under all dosage levels The level 2 to 8% before being administered, reaches peak value in 24-48 hours after SC dosage.

Corrected PK concentration is for analyzing following pharmacokinetic parameter, the area under the curve including concentration relative time (AUC) and in Phoenix WinNonlin software the bioavilability of non-compartmental analysis (NCA) method is used.For s.c. The equal standardized part AUC of dosage is administered from 0-336 hours calculating bioavilabilities with IV, and it is for group 1 Higher than baseline 14.12% to 102%, for group 2%, it is higher than baseline 10.1% to 35.5% (referring to table 8A).

Table 8A: bioavilability

Group 1 (single 25IU/kg)

Area (AUC) under average value -27.4AUCSC0-336: subcutaneous administration 0 to 336 hour corrected partial trace (CSL654_3003 research)

Area (AUC) (CSL654_3001 under 0 to 336 hour corrected partial trace is administered in AUCIV0-336:IV Research)

* receive 50IU/kg IV dosage, calculate bioavilability with dose normalized AUC

Group 2 (single 50IU/kg)

Pharmacokinetic analysis

It is modeled and is simulated using Phoenix WinNonlin software.For the building of data expression and figure, use Excel and R software.

Using simple method for congregating (Pooled approach) compartment medicine is carried out for dynamic to each individual subjects Mechanical analysis.Initial estimation is obtained from NCA.Using compartment model be fitted estimation volume of distribution (V), clearance rate (CL) and Absorption rate constant (Ka).

Gradually model selection is based on the minimum of target function value (OFV).It (such as is observed by various diagnostic graphs (DV), (IPRED) relative time of individual prediction) inspection model appropriateness and performance.

Final mask is a Room additive errors model, is further used for simulating.Provide each subject's in table 8B Estimates of parameters.

Due to being up to 336 hours limited sampling windows, 4 time points are increased after 336 hours to estimate to eliminate, Assuming that the index decreased of FIX activity level similar with IV.By this it is assumed that the practical SC score absorbed is likely larger than current mould The score estimated in type fitting/simulation.

Table 8B: the parameter Estimation from a Room additive model

Group 1 (single 25IU/kg)

Parameter	38	39	40	50
					Clearance rate (dL/hr)	2.11	1.08	0.77	0.89
Volume (dL)	56.09	140.06	24.04	2.53
					Ka(1/hr)	0.0037	0.0057	0.0025	0.0046

Group 2 (single 50IU/kg)

Parameter	38	39	16	21
					Clearance rate (dL/hr)	3.00	1.76	1.92	1.96
Volume (dL)	45.95	37.71	9.98	97.85
					Ka(1/hr)	0.0047	0.0029	0.0032	0.0042

Repeated doses analyze (every 3 days 25IU/kg)

For group 3, previous at least 14 days and in application 25IU/kg rIX-FP agent in 24 hours after rIX-FP application Amount, before analyzing the administration of blood sample, 30 minutes after application, 3,8,24,48 and 72 hours plasma F IX activity and blood coagulation it is living Change.

Subject in group 3 returns to study site to carry out the SC dosage 2 and 3 of 25IU/kg rIX-FP.In SC dosage 3 Before, the plasma F IX activity of blood sample is analyzed.SC dosage 4 to 12 is applied at home and returns to study site within subject every 3 days Carry out SC dosage 13.Before SC dosage 13, also collection blood sample is by for plasma F IX activity.In study site, application is every After a dosage, researcher assesses local tolerance (at 30 minutes) and records AE and any adjoint treatment.Dosage 14 is also in Middle application.

Give the SC dosage 15 of 25IU/kg rIX-FP in study site, and before administration, application after 30 minutes, 3,8, 24, the plasma F IX activity of blood sample and the activation of blood coagulation test are assessed within 48 and 72 hours.It is every for being given in study site A dosage carries out local tolerance and the assessment of AE and any concomitant therapy.

The FIX activity of each time point of each patient is shown in table 9 and Fig. 7.

Table 9: the FIX activity observed in the patient of every 3 days application 25IU/kg in group 3

Pharmacokinetic analysis

Program 7.3 editions for this analysis (ICON Development Solutions, Ellicott City, Maryland, USA) [NONMEM users' guidebook].Pirana (version 2 .8.1) is used as NONMEM interface.Use First- Order Conditional Estimation Method with Interaction (FOCEI) estimates PK parameter.Perl- Speaks-NONMEM (PsN, version 3 .5.3) is executed for NONMEM and VPC.R (version 2 .14.1) (http://r- project.org) it is used for the post-processing and drawing of result.

The data from research CSL654_2001, CSL654_2004, CSL654_3001, CSL654_3002 will be used FIX from CSL654 is active previously developed from rIX-FP (room 2-) group PK model intravenously applied (Idelvion final mask) is used as the starting point of this analysis.Configuration parameter is indicated with the parameter of clearance rate and volume of distribution.First Preceding modeling has shown that may be advantageous using log transformed data；Therefore, the FIX activity of Logarithm conversion is explored.Make While implementing FIX activity after the intravenous and subcutaneous route of rIX-FP application with the Advan4Trans4 model library in NONMEM Fitting.

For FIX activity PK model, it is assumed that real endogenous FIX activity level no more than 2%FIX activity, with FIX It is consistent that standard is included in the research of activity≤2%.Therefore, when the FIX that subject observes before the rIX FP of the first dosage lives Property value be greater than 2% when, it is believed which reflects the remnants of the previous FIX product other than real endogenous FIX activity level Contribution.Thus, it is seen that FIX activity level (FTOT) be assumed endogenous baseline level (FEND), previous FIX product appoint What residual contribution (FPP) and the FIX level for being attributed to CSL654 application increase the summation of (FEX), as shown in following equation:

FTOT=FEX+FEND+FPP

Wherein:

FPP=BSE*EXP (- (CL/V1) * TIME) is but if BSE < 2 item FPP=0.

FIX activity before the administration that BSE=is observed

When FPP drops below the minimum of following values, then FPP=0:BSE, screens FIX activity or 2%.

Covariant modeling: in the analysis, weight is considered as significant covariant.

Model evaluation: pass through the goodness of fit (GoF) of various charts and the metric evaluation model of calculating:

The figure relative to group and individual prediction concentrations observed；

Condition weighted residual (CWRES) is relative to Prediction concentration and relative to the figure of time；

Final mask be fitted simultaneously group 1 and group 3 from 3003 researchs SC data and from including 2001, 2004,2 Room models of the IV data of 3001,3002 and 3003 research.Weight is that central volume is related with weight adjustment dosage Significant covariant, and weight is the related significant covariant of clearance rate.

Simulation:

The FIX that final FIX activity group PK model is used to simulate following IV administration scene in teenager/Adult group is living Property-time graph (FTOT and FEX):

The steady dose of daily 25IU/kg

The steady dose of every 2 days 25IU/kg

The steady dose of every 3 days 25IU/kg

For these simulations, the FIX activity level and from packet before analogue data is concentrated using the administration observed of individual Teenager/adult whose body weight in group PK modeling is included, and assumes 10 minutes infusion durations.For each mould Quasi- scene, executes the repetition of 1000 simulated data sets.

The valley FIX activity level simulated and observed is summarised in table 10.

The valley FIX activity level (IU/dL) that table 10: simulating and observes

Fig. 8 A-C shows active (IU/dL) phase of the FIX of the 25IU/kg of daily, every two days and time interval every three days To the simulation drawing of time, and Fig. 8 D shows the repetition PK data observed with the 15th dosage from group 3 (n=3) The simulation drawing of active (IU/dL) relative time of the FIX of every 3 days 25IU/kg of covering.

It summarizes:

FIX activity data (intermediate value 6.2IU/dL) card observed after every 3 days 25IU/kg (repeating subcutaneous rIX-FP administration) Be illustrated this rIX-FP administration route be able to maintain that patient FIX activity level in slight haemophiliac's subclass (for example, > 5IU/dL FIX is horizontal).Group's FIX PK model of revision includes and sufficiently characterizes the subcutaneous route of rIX-FP application.rIX- The single of FP and the individual patient FIX PK fitting after repetition subcutaneous administration demonstrate the effectiveness of final mask.Simulation confirms observation To subcutaneous rIX-FP PK data be aligned well with the PK curve of prediction.

Claims

1. a kind of fusion protein, it includes

A) people's factors IX (FIX), and

B) human albumin

It is used in the method for its bleeding for being used to prevent in preventive administration scheme human patient, wherein people FIX passes through peptide linker It being connect with the N-terminal of human albumin, the peptide linker can be cut by participation blood coagulation or by the protease of activated by thrombin, and Wherein the fusion protein is waited for the dosage of about 10-50IU/kg, using about once a day to dosing interval skin about once a week Under be applied to the subject.

2. the fusion protein used according to claim 1, wherein the dosage is about 10IU/kg.

3. the fusion protein used according to claim 1, wherein the dosage is about 25IU/kg.

4. the fusion protein used according to claim 1, wherein the dosage is about 50IU/kg.

5. the fusion protein used according to claim 1, wherein the dosing interval be about once a day, or it is about secondary on every Thursdays, Or it is 2 days about every or 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.

6. the fusion protein used according to claim 2, wherein the dosing interval be about once a day, or it is about secondary on every Thursdays, Or it is 2 days about every or 3 days about every, or about twice a week.

7. the fusion protein used according to claim 3, wherein the dosing interval be about once a day, or it is about secondary on every Thursdays, Or it is 2 days about every or 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.

8. the fusion protein used according to claim 4, wherein the dosing interval be about once a day, or it is about secondary on every Thursdays, Or it is 2 days about every or 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.

9. the fusion protein used according to claim 3, wherein the dosing interval is about once a day.

10. the fusion protein used according to claim 3, wherein the dosing interval is 2 days about every.

11. the fusion protein used according to claim 3, wherein the dosing interval is 3 days about every.

12. the fusion protein used according to claim 1, wherein the dosage is about 25IU/kg, the dosing interval is about every It is primary, and total FIX activity trough level in blood plasma maintains about 8 to about 34IU/dL.

13. the fusion protein used according to claim 1, wherein the dosage is about 25IU/kg, the dosing interval is about every It is primary, and total FIX activity trough level in blood plasma maintains about 17IU/dL.

14. the fusion protein used according to claim 1, wherein the dosage is about 25IU/kg, the dosing interval is about every 2 days, and total FIX activity trough level in blood plasma maintains about 4 to about 17IU/dL.

15. the fusion protein used according to claim 1, wherein the dosage is about 25IU/kg, the dosing interval is about every 2 days, and total FIX activity trough level in blood plasma maintains about 8IU/dL.

16. the fusion protein used according to claim 1, wherein the dosage is about 25IU/kg, the dosing interval is about every 3 days, and total FIX activity trough level in blood plasma maintains about 2.5 to about 12IU/dL.

17. the fusion protein used according to claim 1, wherein the dosage is about 25IU/kg, the dosing interval is about every 3 days, and total FIX activity trough level in blood plasma maintains about 5IU/dL.

18. the fusion protein used according to claim 1 to any one of 17, wherein in entire dosing interval, the blood plasma of FIX Level is maintained above the trough level of baseline at least about 1%.

19. the fusion protein used according to claim 1 to any one of 17, wherein in entire dosing interval, the blood plasma of FIX Level is maintained above the trough level of baseline at least about 3%.

20. the fusion protein used according to claim 1 to any one of 17, wherein in entire dosing interval, the blood plasma of FIX Level is maintained above the trough level of baseline at least about 5%.

21. the fusion protein used according to claim 1 to any one of 17, wherein in entire dosing interval, the blood plasma of FIX Level is maintained above the trough level of baseline at least about 10%.

22. the fusion protein used according to claim 1 to any one of 17, wherein in entire dosing interval, the blood plasma of FIX Level is maintained above the trough level of baseline at least about 15%.

23. the fusion protein used according to claim 1 to any one of 22, wherein the connector can by FIXa and/or FVIIa/ tissue factor (TF) cutting.

24. the fusion protein used according to claim 23, wherein the connector includes to be selected from SEQ ID NO:1 and SEQ ID The sequence of NO:2.

25. the fusion protein used according to claim 23 or 24, wherein the sequence of the fusion protein and SEQ ID NO:3 institute The sequence shown has at least 70% identity.

26. the fusion protein used according to claim 23 or 24, wherein the sequence of the fusion protein has SEQ ID NO:3 Shown in sequence.

27. the fusion protein used according to claim 1 to any one of 26, wherein the human patient suffers from hemophilia B.

28. the fusion protein used according to claim 1 to any one of 27, wherein providing the fusion protein for about 100 to 400IU/ml concentration application.

29. the fusion protein or method that are used according to claim 28, wherein providing the fusion protein for about 100,200 Or the concentration application of 400IU/ml.

30. a kind of method for the bleeding for preventing human patient in preventive administration scheme comprising Xiang Suoshu subject is with about every It is once to the fusion protein of dosing interval subcutaneous administration about 10-50IU/kg dosage about once a week, the fusion protein packet Contain

A) people's factors IX (FIX), and

B) human albumin,

Wherein people FIX is connected by the N-terminal of peptide linker and human albumin, and the peptide linker can be by participation blood coagulation or by blood coagulation The protease of enzyme activation is cut.

31. method according to claim 30, wherein the dosage is about 10IU/kg.

32. method according to claim 30, wherein the dosage is about 25IU/kg.

33. method according to claim 30, wherein the dosage is about 50IU/kg.

34. method according to claim 30, wherein the dosing interval be about once a day, it is or about secondary on every Thursdays, or about every 2 It, or it is 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.

35. according to the method for claim 31, wherein the dosing interval be about once a day, it is or about secondary on every Thursdays, or about every 2 It, or it is 3 days about every, or about twice a week.

36. according to the method for claim 32, wherein the dosing interval be about once a day, it is or about secondary on every Thursdays, or about every 2 It, or it is 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.

37. according to the method for claim 33, wherein the dosing interval be about once a day, it is or about secondary on every Thursdays, or about every 2 It, or it is 3 days about every, or about twice a week, or it is 5 days about every, or about once a week.

38. according to the method for claim 32, wherein the dosing interval is about once a day.

39. according to the method for claim 32, wherein the dosing interval is 2 days about every.

40. according to the method for claim 32, wherein the dosing interval is 3 days about every.

41. method according to claim 30, wherein the dosage is about 25IU/kg, the dosing interval be about once a day, And total FIX activity trough level in blood plasma maintains about 8 to about 34IU/dL.

42. method according to claim 30, wherein the dosage is about 25IU/kg, the dosing interval be about once a day, And total FIX activity trough level in blood plasma maintains about 17IU/dL.

43. method according to claim 30, wherein the dosage is about 25IU/kg, the dosing interval is 2 days about every, and And total FIX activity trough level in blood plasma maintains about 4 to about 17IU/dL.

44. method according to claim 30, wherein the dosage is about 25IU/kg, the dosing interval is 2 days about every, and And total FIX activity trough level in blood plasma maintains about 8IU/dL.

45. method according to claim 30, wherein the dosage is about 25IU/kg, the dosing interval is 3 days about every, and And total FIX activity trough level in blood plasma maintains about 2.5 to about 12IU/dL.

46. method according to claim 30, wherein the dosage is about 25IU/kg, the dosing interval is 3 days about every, and And total FIX activity trough level in blood plasma maintains about 5IU/dL.

47. according to the method for any one of claim 30 to 46, wherein the blood plasma level of FIX is tieed up in entire dosing interval Hold the trough level higher than baseline at least about 1%.

48. according to the method for any one of claim 30 to 46, wherein the blood plasma level of FIX is tieed up in entire dosing interval Hold the trough level higher than baseline at least about 3%.

49. according to the method for any one of claim 30 to 46, wherein the blood plasma level of FIX is tieed up in entire dosing interval Hold the trough level higher than baseline at least about 5%.

50. according to the method for any one of claim 30 to 46, wherein the blood plasma level of FIX is tieed up in entire dosing interval Hold the trough level higher than baseline at least about 10%.

51. according to the method for any one of claim 30 to 46, wherein the blood plasma level of FIX is tieed up in entire dosing interval Hold the trough level higher than baseline at least about 15%.

52. according to the method for any one of claim 30 to 51, wherein the connector can be organized by FIXa and/or FVIIa/ The factor (TF) cutting.

53. according to the method for claim 52, wherein the connector includes the sequence selected from SEQ ID NO:1 and SEQ ID NO:2 Column.

54. according to the method for claim 52 or 53, wherein sequence shown in the sequence of the fusion protein and SEQ ID NO:3 With at least 70% identity.

55. according to the method for claim 52 or 53, wherein the sequence of the fusion protein has sequence shown in SEQ ID NO:3 Column.

56. according to the method for any one of claim 30 to 55, wherein the human patient suffers from hemophilia B.

57. according to the method for any one of claim 30 to 56, wherein provide the fusion protein for about 100 to The concentration of 400IU/ml is applied.

58. according to the method for claim 57, wherein providing the fusion protein for the dense of about 100,200 or 400IU/ml Degree application.